During the past year, this laboratory continued studies on the mechanism of HIV entry, and on the development of novel protective and treatment strategies based on the molecules involved in entry. 1) Molecular mechanisms of HIV Env-mediated entry/fusion. a) Effects of synthetic peptides based on the extracellular loop of CCR5 on Env function. We found that peptides from each of the extracellular loops inhibited fusion and infection mediated by CCR5-using Envs, but not by Envs that cannot use CCR5. Several lines of evidence indicated that the peptides act by binding to Env. b) Design of a soluble CCR5 mimic. We have initiated collaborative efforts to design a soluble protein containing the extracellular loops of CCR5, with the hope that it will bind to the CCR5-binding region of gp120. 2) Novel anti-HIV agents based on HIV Env/receptor interactions. a) sCD4-17b, a potent HIV-1 neutralizing agent. sCD4-17b is a recombinant chimeric protein containing soluble CD4 attached via a long flexible polypeptide linker to a single chain antibody that binds to a CD4-induced epitope of gp120 involved in binding to coreceptor. While the protein neutralizes some HIV-1 strains with extremely high potency, other strains appear resistant, despite the known presence of binding sites for CD4 and 17b. We hypothesize that the resistance of these isolates reflects structural features of the corresponding gp120 molecules (V1/V2 and V3 loops, glycosylation) that renders the original linker (35 residues) insufficiently long to enable simultaneous binding of the CD4 and 17b moieties. We have engineered sCD4-17b variants with longer linker lengths, and prepared corresponding recombinant baculoviruses for high level expression. We have also continued efforts to express sCD4-17b in Lactobacillus, with the idea of colonizing the vaginal tract to provide a means for protection against sexual transmission that is durable, economically feasible, and controlled by women. b) Anti-HIV immunotoxin. NIH has licensed 3B3-PE38 to IVAX, to produce clinical grade material for Phase I trials. We have also initiated a collaboration to test the efficacy of anti-HIV immunotoxins in SHIV-infected rhesus macaques.